Microfabricated tissue as a substrate for pigment epithelium transplantation

ABSTRACT

An ocular implant is provided with a substrate and a membranous tissue layer secured to the substrate. Cells such as IPE cells, RPE cells and stem cells are attached on the surface of the membranous tissue layer either in situ or in vivo through cells transplantation. The cells are separated into regions on the surface by creating a pattern on the surface enclosing regions for receiving the cells. The substrate is a bioabsorbable and/or polymeric substrate. Examples of membranous tissue layer are lens capsule, inner limiting membrane, corneal tissue, Bruch&#39;s membrane tissue, amniotic membrane tissue, serosal membrane tissue, mucosal membrane tissue and neurological tissue. The membranous tissue layer could have a micropattern of biomolecules. A microfluidic network could be placed onto the microfabricated membranous tissue layer.

FIELD OF THE INVENTION

The present invention relates generally to the field of treatment of eyedisorders, in particular retinal disorders such as age-related maculardegeneration, retinitis pigmentosa, and other retinal diseases. Inaddition, the invention relates to methods and apparatus for modifyingtissues, and for the transplantation of cells and tissues.

BACKGROUND OF THE INVENTION

Diseases of the retina, such as age-related macular degeneration (AMD),retinitis pigmentosa (RP), and other diseases, are the leading cause ofsevere visual impairment or blindness in the industrialized world. Onehallmark of AMD, as in RP, is the degeneration and loss of cells of theretinal pigment epithelium (RPE). Bruch's membrane is also thought to bedamaged; such damage may be the initiating stimulus for RPE demise. RPEcells are vital to the survival and proper functioning of retinalphotoreceptors, which are the only cells in the eye which directly senselight. RPE degeneration in retinal diseases such as AMD and RP isrelated to the loss of photoreceptor function and the visual impairmentthat is associated with these diseases.

The RPE is located adjacent to the neural retina, directly opposed tothe retinal photoreceptors. RPE cells in vivo form one cell thickcobblestone-like tissue linked together by tight junctions, withdifferentiated apical and basal membranes. The RPE cells in vivo growtightly packed together at high density to form a tight epithelium thatacts as a barrier regulating transport between the photoreceptors andthe underlying Bruch's membrane, choroid and the choroidal vasculature.The apical portion of the RPE is adapted to surround and engulfphotoreceptor outer segments, to perform its vital functions ofphagocytosis and digestion of shed photoreceptor tips, and of recyclingretinal for re-use in photopigments. The basal portion of the RPE isapposed to Bruch's membrane, a highly vascularized supporting membranewhich supplies the RPE and photoreceptors with needed oxygen andnutrients, and prevents the accumulation of carbon dioxide and otherwaste products which would otherwise impair retinal function. Damage toBruch's membrane, which may occur due to accumulation of waste productsfrom outer segment metabolism, for example, prevents the exchange ofoxygen, growth factors and waste products. Such impaired exchange leadsto hypoxia in the photoreceptors. In response, it is thought thatsurvival signals are sent out to initiate the in-growth of neovascularvessels, and so to the wet form of AMD.

The iris pigment epithelium (IPE), which, like the RPE is derived fromthe neuroectoderm of the embryo, is located adjacent to the iris at thepart of the eye opposite to the retina. Thus, in place in the intacteye, IPE cells are remote from retinal photoreceptors. Although muchabout IPE cell physiology and function remains unknown, like RPE cells,IPE cells in culture have been shown to be capable of phagocytosis ofphotoreceptor outer segments. RPE cells may be grown on artificialsubstrates (Pfeffer, B. A., Chapter 10, “Improved Methodology for CellCulture of Human and Monkey Retinal Pigment Epithelium,” Progress inRetinal Research, Vol. 10 (1991) Ed. Osborn, N., and Chader, J.; Lu etal., J. Biomater. Sci. Polymer Edn. 9:1187-1205 (1998), and Lu et al.,Biomaterials 20:2351-2361 (1999). In addition, there have been attemptsto use lens capsule tissue as a substrate for growing RPE and IPE cells(Hartman et al., Graefe's Archiv Clin Exp Ophthalmol 237:940-945 (1999);Nicolini et al., Acta Ophthalmol Scand 2000 October;78(5):527-31)).

Many approaches have been tried in the treatment of degenerative andprogressive retinal diseases. For example, attempted treatments for AMDinclude photodynamic therapy, radiation therapy, and macular relocationin order to repair, retard the progression, or compensate for theeffects of the disease. However, such approaches have not met with greatsuccess.

Since RPE cell loss occurs in many retinal diseases, the transplantationof cells has great attraction as a therapy and possible cure for AMD andother diseases. Direct transplantation of RPE cells into the retina hasbeen attempted in order to replace lost RPE cells. However, thisapproach has not succeeded in the past, due in part to the failure ofthe transplanted cells to function properly and in part due to rejectionof the cells by the host animals.

Transplantation of RPE cells has been suggested as a therapy for retinaldystrophy (U.S. Pat. No. 5,962,027 to Hughes and U.S. Pat. No. 6,045,791to Liu). All patents and publications named herein, both supra andinfra, are hereby incorporated by reference in their entirety. Inaddition, experimental evidence that IPE cells could substitute for RPEcells has led to preliminary attempts to transplant IPE cells in animalsand in order to ameliorate symptoms of AMD (Abe et al., Tohoku J. Exp.Med. 189:295-305 (1999), Abe et al., Cell Transplantation 8(5):501-10(1999); Schraermeyer et al., Invest. Opth. Vis. Sci. 40(7):1545-56(1999); Thumann et al., Transplantation 68(2)195-201 (1999); Abe et al.,Tohoku J. Exp. Med. 191:7-20 (2000); Abe et al., Current Eye Research20(4):268-275 (2000); Lappas et al., Graefes 's Arch Clin Exp Ophthalmol238:631-641 (2000), Thumann, et al., Arch. Ophthalmol. 118:1350-1355(2000)).

However, challenges to both IPE and RPE transplantation methods includei) difficulty in repairing the diseased Bruch's membrane, ii) inabilityto secure and position newly transplanted cells, and iii) lack ofcontrol over extracellular matrix signaling molecules that are criticalto the structure, function, and survival of the pigment epithelial cell.For these and other reasons, techniques for IPE and RPE transplantationusing antibiotics or immunosuppressants have not been successful. Therehas been no demonstration of significant visual improvement with theseapproaches, and problems of tissue reintegration remain. Thus, despitethe apparent promise of the transplantation approach, AMD and otherretinal diseases remain without successful therapeutic interventions.

Accordingly, there is need in the art for novel methods and apparatusfor modification of tissues for transplantation and for transplantationof such tissues for the relief of retinal diseases.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to methods, apparatus, and relatedproducts for modifying tissues and growing cells for transplantation. Inparticular, the invention is directed towards methods, apparatus, andrelated products for transplantation of cells and tissues into theretina for treatment of retinal diseases such as AMD and RP. Tissuesmodified by the methods disclosed herein are termed microfabricatedtissues. The invention includes microfabricated membranous tissues,including microfabricated ocular membranous tissues, for examplemicrofabricated lens capsule tissues, microfabricated inner limitingmembrane tissues, microfabricated Bruch's membrane tissues, and othertissues. The invention further includes microfabricated membranoustissues for use in transplantation, methods for microfabricatingmembranous tissues, methods for using microfabricated membranoustissues, methods for growing cells on microfabricated membranoustissues, and methods for transplanting microfabricated tissues and cellsinto the eye of an animal. For example, the animal may be a human.

A microfabricated membranous tissue embodying features of the inventionmay be prepared by contacting membranous tissue with a substrateincluding a bioabsorbable material, which may be submersed in phosphatebuffered saline, or by coating a surface of a membranous tissue with abioabsorbable material, and modifying the membranous tissue eitherbefore or after coating or contacting the tissue with the substrate.Suitable bioabsorbable materials include collagen; glycosaminoglycans;chitosan; poly(hydroxyalkanoates); poly(α-hydroxy acids); polyglycolicacid (PGA); polylactic acid (PLA); polylactide-polyglycolide (PGA-PLA)mixtures, alloys and copolymers (PLGA); poly(dioxanones);poly(E-caprolactone); poly(ortho esters); poly(anhydrides);poly(phosphazenes); poly(amino acids); and other compounds, polymers,copolymers, alloys, mixtures and combinations of these compounds.Suitable membranous tissue includes lens capsule, inner limitingmembrane, Bruch's membrane, corneal tissue, amniotic membrane, serosalmembrane tissue, mucosal membrane tissue, and other tissue includingneurological tissue.

A microfabricated membranous tissue, coated with, in contact with, orplaced on a substrate, may further have cells grown upon it, by a methodincluding coating membranous tissue or contacting membranous tissue witha substrate, the tissue optionally being submersed in phosphate bufferedsaline or other physiological solution, modifying the membranous tissue,and applying cells (such as IPE and RPE cells) to the modifiedmembranous tissue. A microfabricated membranous tissue may also bemodified by partly covering the membranous tissue with a stencil andgrowing cells on the exposed surface of the membranous tissue.

Methods for modifying membranous tissues may include mechanical methodsincluding mechanical ablation, mechanical contact, and photoablationmethods. The methods of the invention for modifying membranous tissuesmay be applied to a variety of tissues, including ocular membranoustissues. For example, the methods of the invention include methods formodifying lens capsule tissue, such as human lens capsule tissue, andfor modifying inner limiting membrane tissue, such as human innerlimiting membrane tissue.

Methods for modifying membranous tissues include bulk modificationmethods and surface modification methods. Surface modification methodsand bulk modification methods may be applied alone, or may each beapplied together to the same membranous tissue. Modification of thesurface and bulk properties of the membranous tissue improves thetissue's suitability for transplantation into an animal. Such tissuemodification may improve the ability of cells to attach and grow on thetissue, and may improve the permeability properties of the tissue sothat nutrients, electrolytes, and other desired substances are betterable to pass through the modified tissue.

The methods of the invention, whether bulk or surface modificationmethods, include removal of membranous tissue, such as a lens capsule oran inner limiting membrane, from an eye, flattening the membranoustissue onto a glass or plastic substrate, such as a coverslip, submersedin phosphate buffered saline, or flattening the membranous tissue onto atemporary dissolvable polymer for ease of surgical transplantation. Themodified tissue provides a suitable substrate for cells, and may beexposed to cells which may attach and grow. The modified tissue, withadherent cells if any were applied to and grown on the tissue and/orwith polymer, if any, may next be transplanted into a desired locationwithin the body of an animal. Following transplantation, where themodified tissue has been prepared with a dissolvable polymer, thepolymer will dissolve and be absorbed by the body of the animal intowhich the tissue has been transplanted, leaving the transplanted tissueand cells in place.

Suitable dissolvable polymers include poly-lactic acid, polyglycolicacid, polyorthoesters, poly anhydrides, polyphosphazines, poly-lacticacid glycolic acid copolymers (PLGA), including PLGA (e.g., a 50:50mixture of lactic to glycolic acid copolymer, a 90:10 mixture, or otherproportions), poly-lactic acid polymers (PLLA), polyethyleneglycol/polylactic acid copolymer (PEG/PLA), and blends and co-polymersthereof.

Bulk modification methods are those where substantial portions of themembranous tissue, not limited to surface portions of the tissue, aremodified by the method. Surface modification methods are those where themembranous tissue is modified at and near to the surface, but is notgreatly modified in other portions of the tissue.

Bulk modification methods for modifying membranous tissue, includingocular membranous tissue such as lens capsule tissue and inner limitingmembrane tissue, include methods for modifying the thickness,permeability, and other properties of the tissue. Bulk modificationmethods include mechanical ablation, including rubbing, scraping,cutting, and applying tension, contacting the membranous tissue with acontacting surface such as a stamp, and producing a micropattern in themembranous tissue. In one embodiment of the bulk modification method,treatment after removal and flattening of the membranous tissue includesuse of a laser or ion beam to modify the surface of the membranoustissue to reduce the overall thickness of the tissue. For example, thelens capsule, which can normally be up to about 8 to 14 micrometers (μm)thick, may be ablated by photoablation with an excimer laser to be about2 to 5 μm thick, so that the overall thickness of the altered lenscapsule mimics the thickness of Bruch's membrane (about 2 to 4 μm).

In another embodiment of the bulk modification method, such furthertreatment includes photoablation using a laser, such as an excimer,titanium sapphire, or YAG laser, or ion beam treatment, to producemicropores or pits in the membranous tissue. The micropores may be sizedon the order of a few micrometers or less in diameter. A micropattern ofmicropores or pits produced in the membranous tissue by such treatment.

Membranous tissue may be treated by impregnation with a deactivatedcollagenase enzyme that is activated by laser light illumination. Forexample, very small regions sized less than a micrometer in diameter oftissue may be activated by illumination with a 2-photon confocal lasersystem.

Enzymes may be deposited onto the membranous tissue effective tobiologically etch the surface and interior of the membranous tissueeffective to provide desired topology and surface adhesion properties tothe tissue. In some embodiments of this method, the deposition stepincludes contacting the membranous tissue with a contacting surface,such as a microcontact printing stamp, carrying enzymes effective tobiologically etch the surface and interior of the membranous tissue.

Treatments may include surface modification of the membranous tissue aswell. For example, treatment may include deposition of patterns ofbiomolecules onto membranous tissue, and production of patterns of poresor pits or other surface features by laser or ion beam treatment. Insome embodiments of this method, the patterns are sized on the order ofa few micrometers or less. In other embodiments of this method, thebiomolecules include peptides and small chain polymers effective todeactivate selective cell attachment sites on membranous tissue.

In one embodiment of the surface modification method, microcontactprinting techniques are used to fabricate chemical micropatterns ofbiomolecules on the membranous tissue. Membrane surfaces may also bemodified by mechanical ablation methods including rubbing, scraping andflowing solutions over the surface.

In another embodiment of the surface modification method, the surface ofthe membranous tissue is masked to cover part, but not all, of thesurface of the tissue, and then irradiated with ultraviolet (UV)radiation effective to denature the extracellular matrix (ECM) of theexposed portions of tissue. In order to activate only certain portionsof the surface of the membranous tissue, a deactivating substance suchas polyvinyl alcohol (PVA) or mucilage can be applied to the surface ofthe tissue, and an excimer laser can be used to ablate a micropattern onthe membranous tissue surface through an irradiation mask.

The masking step may be performed by placing a grid onto the surface ofthe membranous tissue, or by using microcontact printing techniques toapply a pattern of protecting molecules onto the surface of themembranous tissue effective to prevent ECM denaturation in regionscovered by the protecting molecules or grid.

Cells may be grown on microfabricated membranous tissues. For example,cells may be applied to microfabricated membranous tissues, either insitu or in vivo through transplantation, which may have patterns ontheir surfaces. In further embodiments, the microfabricated membranoustissue may be lens capsule tissue or inner limiting membrane tissue, andthe cells may be IPE cells. In yet other embodiments of the invention,the microfabricated membranous tissues and the cells may be obtainedfrom the same animal. In this last case, transplantation of the modifiedtissue and cells into that animal would be autologous transplantation,which would not suffer from rejection by the animal's immune system.

The invention also provides methods for using microfabricated membranoustissues, including surgical methods for transplanting microfabricatedmembranous tissues into an animal. The methods for transplantingmicrofabricated membranous tissues into an animal include surgicalmethods for transplanting microfabricated membranous tissues into theeye of an animal, such as transplantation of microfabricated lenscapsule tissue or microfabricated inner limiting membrane tissue near toor into the retina of an animal. The transplanted tissue may furtherinclude cells grown on microfabricated lens capsule tissues ormicrofabricated inner limiting membrane tissues. In preferredembodiments, the transplanted microfabricated membranous tissue includesIPE cells grown on microfabricated lens capsule tissues ormicrofabricated inner limiting membrane tissues, and may be autologoustissue and cell transplants.

The invention also provides products useful in fabricating and usingmicrofabricated tissues. Such products include products and tools formaking modified ocular membranous tissues, including microfabricatedlens capsule and inner limiting membrane tissues, and products and toolsfor transplanting the transplanted tissues and cells into the eye of ananimal.

The present invention is directed to methods and related products fortreating retinal diseases such as AMD, RP, and other retinal diseases.For example, one therapy for AMD is to transplant suspensions of eitherretinal pigment epithelial (RPE) cells, iris pigment epithelial (IPE)cells, stem cells, or other cells, to rescue the diseased retina. Thepresent invention provides novel tissue engineering techniques toprecision engineer autologous human tissues (e.g., human lens capsule)as a substrate for transplanting cells, such as IPE cells, RPE cells,stem cells, and other cells. Transplanted pigment epithelium cells grownon the modified tissue and substrates of the invention are able to growto high density and to exhibit features indicative of differentiation,important characteristics of these cells in normal retinas. In addition,unlike prior methods, the modified membranous tissues (includingmodified ocular membranous tissues, such as lens capsule, inner limitingmembrane, and other substrates provided by the present invention, andsuch substrates with growing epithelial cells) are effective to replacemany of the functions of Bruch's membrane, which may be damaged indegenerative retinal diseases. Thus the present methods and apparatusfor transplantation of pigment epithelial cells provide transplantedcells which grow to high density and are able to perform neededphysiological functions lacking in patients with retinal degenerativediseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a cross-sectional view of microfabricated membranous tissueon a dissolvable substrate embodying features of the invention.

FIG. 1B is a cross-sectional view of an eye having microfabricatedtissue on a dissolvable substrate implanted in the subretinal space ofits eye.

FIG. 1C is a detailed cross-sectional view of the microfabricatedtissue, retina and subretinal space of the eye.

FIG. 2 illustrates a microcontact printing stamp useful for producingmicrofabricated membranous tissue embodying features of the invention.

FIG. 3 illustrates microfabricated lens capsule tissue after contactwith a microcontact printing stamp embodying features of the invention.

FIG. 4 illustrates a poly(dimethylsiloxane) (PDMS) stamp formicropatterning membranous tissue according to methods embodyingfeatures of the invention.

FIG. 5 illustrates cells growing on a human lens capsule micropatternedwith the PDMS stamp illustrated in FIG. 4.

FIG. 6 is a photomicrograph of a microfabricated lens capsule on apoly-lactide/polyglycolide carrier matrix.

FIG. 7 is a photomicrograph of a section of rabbit retina containing amicrofabricated lens capsule on a poly-lactide/polyglycolide carriermatrix one week after implantation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and apparatus for modifyingtissues and cells for transplantation. The methods of the invention formodifying tissues may be applied to a variety of tissues from a varietyof organs. The following definitions are helpful in describing theinvention.

The term “autologous” is used herein to refer to cells or tissuesderived from the same animal as other cells or tissues; thus, withrespect to a tissue, cells are autologous cells when they are derivedfrom the same animal as the tissue is derived from; analogously, thetissue is autologous tissue with respect to the cells when the cells andtissue are derived from the same animal.

The term “biomolecule” is used herein to mean a molecule that has abiological activity. Thus, a biomolecule is one that, when in contactwith a cell or tissue, acts on or affects that cell or tissue.

The term “bulk modification” is used herein to mean the modification ofthe properties of substantial portions of a tissue, where suchmodification is not limited to the surface portions of the tissue.

The term “surface modification” is used herein to mean the modificationof the properties of a tissue at and near to the surface of the tissue.

The term “membranous tissue” is used herein to mean any tissue of ananimal that forms a sheet or sheath; membranous tissue commonly enclosesor delimits a tissue, or divides an organ or tissue into separatecompartments. “Ocular membranous tissue” is used herein to meanmembranous tissue derived from the eye of an animal; lens capsule tissueand inner limiting membrane are examples of ocular membranous tissue, asare corneal membranes, Bruch's membrane, and other membranous tissues ofthe eye.

The term “ablation” is used herein to mean the alteration of a tissue,not necessarily including the reduction in the size or the removal oftissue. As used herein, “mechanical ablation” means alteration,reduction, or removal of tissue by mechanical action, such as scraping,scoring, contacting with a contacting surface (such as a stamp),applying tension, or other mechanical method. As used herein,“photoablation” means irradiation by ultraviolet light, laser light, orother radiation, such as by light from an excimer, titanium sapphire,YAG or other laser, effective to alter the surface or bulk properties ofa tissue. “Ion ablation” is used herein to refer to surface or bulkmodification effected by ion beam treatment of a membranous tissue.

A “proteolytic enzyme,” or a “protease,” is a type of molecule that iseffective to at least partially digest (cut into pieces) a protein orpeptide molecule. Examples of proteases and proteolytic enzymes include,but are not limited to, collagenase, trypsin, chymotryptsin, dispase,liberase, thermolysin, pepsin, and papain.

The term “transplantation” is used herein to mean the insertion,deposition or positioning of cells or tissues into an animal. Thedeposition of cells growing on modified lens capsule tissue into thesubretinal space is an example of transplantation.

The term “microcontact printing” is used herein to mean deposition ofdesired molecules onto a surface in a pattern with features sized on theorder of several tens of micrometers or smaller.

The term “microfabrication” is used herein to mean the production ofmodified tissues by surface modification, bulk modification, or both.

The term “microfabricated tissue” is used herein to mean a tissue thathas been altered or modified by microfabrication methods.

A “contacting surface” is a surface configured for contacting a secondsurface and for depositing molecules initially present on the contactingsurface onto the second surface. A “stamp,” “microfabrication stamp,”“microcontact printing stamp,” “microcontact stamp,” or“microfabrication printing stamp” is a contacting surface, and the terms“stamp,” “microfabrication stamp,” “microcontact printing stamp,”“microcontact stamp,” and “microfabrication printing stamp” are usedherein to mean a device adapted to deposit desired molecules in apattern with features sized on the order of several tens of microns orsmaller.

The term “micropattern” is used herein to mean a pattern, such as anordered array, design or contour with features sized on the order ofseveral tens of microns or smaller.

By “dissolvable polymer” is meant a polymer that is biodegradable, andthat upon introduction into an animal may at least partially dissociateand disperse into fluids and tissues of the animal.

A “laser” may be an excimer laser, a titanium sapphire laser, anyttrium-aluminum-garnet (YAG), or other laser. A laser is capable ofemitting a powerful beam of coherent light produced by lightamplification within the laser cavity or crystal of the laser.

As used herein “excimer laser” means a laser light source that provideslaser light of a wavelength below about 400 nanometers (nm). Excimerlasers may be xenon, krypton, or fluorine lasers, or, more preferablymay be an argon fluoride laser. An argon fluoride laser provides laserlight in the ultraviolet, typically with a wavelength of about 193 nm,suitable for ablation of epithelial, connective, and other tissues. Foruse in tissue modification, such as tissue ablation, laser light may bepulsed at between about 1 to 50 Hz with each pulse having a duration ofbetween about 1 to 200 nanoseconds (ns), preferably between about 10 to20 ns. Laser beams, such as produced by argon fluoride lasers, aretypically sized on the order of a few millimeters to several tens ofmillimeters.

A titanium-sapphire (TiS) laser is a tunable laser capable of emitinginfra-red laser light with wavelengths ranging from about 700 to about1100 nm.

An yttrium-aluminum-garnet (YAG) laser, such as a neodimium YAG, ahoronium YAG, or an erbium YAG laser, is a solid-state laser emittinglaser light at a wavelength on the order of a micron. Water moleculesabsorb energy at micron wavelengths; water preferentially absorbs energyat wavelengths near 3 μm, and erbium-doped YAG lasers emit light with awavelength of 2.94 μm, making them particularly suitable for use inphotoablation by rapid, local vaporization of water present in cells andtissues, causing rapid expansion and ablation of tissue.

An ion beam is a beam of ionized gas molecules, typically excited byradio-frequency energy and directed at a target. Ion beam sources usedin the practice of the present invention may be of any kind; an ion beamsource is described, for example, in U.S. Pat. No. 5,216,330 to Ahonen.Ion beams may be used to create holes in materials. U.S. Pat. No.6,093,445 to Nawate describes an ion beam method for producingrectangular and circular holes sized from about 10 nm to about 2 μm.

A tissue implant 10 having microfabricated lens capsule tissue 12 withattached cells 14 and a dissolvable substrate 16 is shown incross-section in FIG. 1A. Cells 14 are attached to and growing uponupper surface 18 of the microfabricated lens capsule tissue 12. Lowersurface 20 of the microfabricated lens capsule tissue 12 is in contactwith the dissolvable substrate 16. Cells 14 are iris pigment epithelialcells, which have apical 22 and basal 24 membranes, with basal membranes22 in contact with upper surface 18 of the microfabricated lens capsule12. Expression of the proper cellular differentiation into basal 22 andapical 24 membranes, as is found in pigment epithelial cells in vivo, isindicative of the proper functioning of the epithelial cells growing onthe microfabricated lens capsule.

FIG. 1B illustrates, in cross-section, an eye 26 of a mammalian animalinto which the tissue implant 10 has been surgically placed. FIG. 1C isa detail of the region within circle 33 of eye 26 including neuralretina 28 and tissue implant 10. Shown in FIGS. 1B and 1C are the neuralretina 28, the iris pigment epithelium (IPE) 30, the retinal pigmentepithelium (RPE) 32 growing on Bruch's membrane 34 which separates thechoroid 36 from the basal membrane 38 of the RPE. The apical membrane 40of the RPE has numerous processes, which enfold and surround thelight-sensitive portions of the photoreceptors in the photoreceptorlayer 42 of the neural retina 28. The space between the apical membranesof the RPE 40 and the photoreceptors 42 is the subretinal space 44. Thechoroid 36 serves to maintain an environment capable of supporting thehigh metabolic demands of the photoreceptor layer 42 in particular andthe neural retina 28 in general by allowing the passage of nutrients andelectrolytes to, and removal of waste products from, the subretinalspace 44.

In a healthy eye, the subretinal space 44 is only a virtual space, therebeing only minimal separation between photoreceptors 42 and apicalportions of the RPE 40. However, in many eye disorders, such as retinaldetachment, the photoreceptors 42 may become separated from the apicalRPE membranes 40. In addition, the neural retina 28 and the pigmentepithelium 30 and 32 may be artificially separated during eye surgery ifdesired. As shown in FIGS. 1B and 1C, a tissue implant 10 may be placedinto the subretinal space 44.

Microfabricated lens capsule 12 with adherent IPE cells 14 is shownimplanted into eye 26 where the IPE cells 30 are able to contactphotoreceptors 42 and provide metabolic support. Dissolvable substrate16 is shown between the lower surface 20 of the microfabricated lenscapsule 12 and the choroid 36, as it is initially after placement of thetissue implant 10. However, the dissolvable substrate 16 will dissolveand be removed from the subretinal space 44 leaving only thetransplanted microfabricated lens capsule 12 and attached IPE cells 14in place in the subretinal space 44.

FIG. 2 is a scanning electron micrograph (SEM) of apoly(dimethylsiloxane) (PDMS) microfabrication stamp embodying featuresof the invention. The grid-lines are about 5 μm wide and are separatedby about 50 μm. The structural height of the PDMS stamp is 7 μm from thebase to the face. Grid lines may be coated with compounds, for example,poly-L-lysine, for placement onto a surface by contacting the surfacewith the stamp. The hexagonal shape was designed to mimic that of RPEcells in the human macula.

FIG. 3 is a SEM of a microfabricated human lens capsule tissue aftercontact with the microfabrication stamp shown in FIG. 2 that had beencoated with a mixture of 2% polyvinyl aclohol (PVA) and 0.1 mg/mLfluorescein. The bright areas show microprinting of the fluoresceinsolution. Micropattern lines follow the same pattern and spacing as thestamp that produced them by contact with the lens capsule surface.

FIG. 4 shows a SEM of a PDMS stamp that has a stamp surface with atopology given by an array of circular wells. Thus, the stamp surface ofstamp has circular depressions that will not receive a coating, whilethe rest of the stamp surface does receive a coating of molecules thatmay then be transferred to any surface with which it becomes in contact.Coating the stamp with molecules, such as PVA, mucilage, or otherinhibitory molecules, and then placing the stamp in contact with asurface, such as a lens capsule surface, leaves a pattern of thosemolecules on the surface everywhere but on the circles themselves. Sucha micropattern of inhibitory molecules allows cells growing on thesurface to attach only on these circular areas. Because the unmodifiedlens capsule surface actively allows growth of cells adherent to it,inhibitory patterns are required for patterned growth. Each circle isabout 50 μm in diameter.

FIG. 5 is a SEM of a human lens capsule that has received a micropatternof PVA inhibitory molecules from the PDMS stamp as illustrated in FIG.3. The scale bar is 25 μm. It is evident that RPE cells are growing in apattern determined by the micropattern deposited on lens capsule bystamp. Cells remained viable and in this pattern for as long as 24 days.It was found that RPE cells on microprinted lens capsule after 24 daysin culture maintained the same pattern after this 24 days, were stillviable, and retained their cuboidal structure. By controlling the widthof the grid lines, cells can be separated to a greater or lesser degree.Thinner grid lines allow growing cells to touch each other, allowingformation of a transporting epithelial layer from the contacting,growing cells.

Attachment of cells onto the microfabricated tissue substrate may bespeeded or enhanced by placement of the microfabricated tissue within aflat-bottomed centrifuge tube along with cells to be grown on themicrofabricated tissue. Centrifugation at low speed, such as, forexample, between about 5,000 to about 15,000 revolution per minuterapidly deposit the cells onto the microfabricated tissue and aid thedirected growth of deposited cells onto the microfabricated tissue.

Placement of microfabricated tissue onto, or coating a microfabricatedtissue with, a carrier matrix aids in its processing and in itsimplantation into the body of an animal. Microfabricated tissue may becoated on one side only, or, in some embodiments of the invention,microfabricated tissue may be coated on both sides. The processing andimplantation of microfabricated lens capsule, microfabricated innerlimiting membrane, microfabricated Bruch's membrane, or othermicrofabricated membranous tissue may be aided in this way; for example,a carrier matrix makes microfabricated tissues more rigid and easier tohandle. In addition, a carrier matrix is effective to prevent foldingand curling of the tissue, allowing implantation of a flat, spread-outtissue sheet. Such a spread-out configuration provides maximal surfacearea for growth of implanted cells, and provides the implanted cellswith maximal access to fluids and surrounding tissues. Biodegradablecarrier matrices embodying features of the invention are flexible,fitting easily to the contours of the retina. Preferably, the carriermatrix is biodegradeable, and so may be resorbed by the host body withina desired time period after placement in the eye. A desired time may beabout a week to a few months, preferably a few weeks to about twomonths, more preferably a carrier matrix embodying features of theinvention biodegrades after implantation in a retina within about twoweeks to about six weeks.

Biodegradable matrix materials suitable for assisting in the processingof tissues and in the implantation of tissues into the eye include, forexample: collagen; glycosaminoglycans; chitosan;poly(hydroxyalkanoates); poly(oc-hydroxy acids), including but notlimited to polyglycolic acid (PGA), polylactic acid (PLA), andpolylactide-polyglycolide (PGA-PLA) mixtures, alloys and copolymers(PLGA); poly(dioxanones); poly(ε-caprolactone); poly(ortho esters);poly(anhydrides); poly(phosphazenes); poly(amino acids); and othercompounds, polymers, copolymers, alloys, mixtures and combinations ofthese materials. FIG. 6 shows a lens capsule (stained blue) on a carriermatrix of poly-lactide/polyglycolide. The scale bar is 1 mm in length.Carrier matrix substrates and coatings may be dyed (e.g., with trypanblue or rhadamine), improving visualization of the tissue to beimplanted during implantation surgery. Such coatings and substrates maybe used for lens capsule, inner limiting membrane, Bruch's membrane, andother membranous tissue, including corneal tissue, amniotic membrane,serosal membranes, mucosal membranes, and neurological tissue.

FIG. 7 shows a section of rabbit retina containing a human lens capsuleon a poly-lactide/polyglycolide carrier matrix. The retinal sectionshown was taken one week after implantation of the lens capsule tissuein the subretinal space between the neural and pigmented retinal cellsin a rabbit eye. The lens capsule has a flat configuration, showing nofolding or curling that would interfere with the flow of nutrients andwaste products to and from the transplanted cells.

Tissues to be modified may be obtained by means known in the art, suchas excision, biopsy, at surgery or at autopsy. As will be understood bythose of ordinary skill in the art, care should be taken to avoid damageor contamination of the membranous tissue during procedures forobtaining it, as by following standard sterile operating procedures. Itwill be understood that the methods and apparatus are suitable formodifying any membranous tissue, including but not limited to ocularmembranous tissue.

In the following, methods and apparatus for modifying tissue aredescribed using primarily lens capsule tissue as exemplary membranoustissue. The methods and apparatus are thus also suitable for modifyinginner limiting membrane tissues and other tissues, and may be used tomodify inner limiting membrane and other tissues as well. The tissuemodification provided by the methods of the invention is effective toalter the properties of the subject tissue to provide a more favorablesubstrate for cell attachment and growth, and to alter the physical andbiochemical properties of the lens capsule tissue to allow more readyexchange of fluid and solutes across the tissue.

Membranous tissue such as lens capsule tissue and inner limitingmembrane may be obtained from donor eyes, or from the patient(autologous tissue) by techniques known in the art, such as followinglens extraction for cataract surgery. For example, lens capsule tissuemay be obtained from an eye after a cataract incision has been made(either a scleral incision or a corneal incision). In this method,viscoelastic is next placed in the anterior chamber following making anincision. The viscoelastic is usually either Healon® (Pharmacia,Kalamazoo, Mich.) or Viscoat® (Alcon, Fort Worth, Tex.). The capsulotomyis then performed by using a cystotome needle. This needle is used topuncture the anterior capsule centrally, creating a capsule flap. Thisflap is then raised using the cystotome needle. Utrata forceps are usedto grasp the flap of the capsule and it is pulled in a circular fashion.Pulling of the capsule for 360° in a controlled fashion will result in around continuous capsulorhexis, exposing the cataract. The lens and lenscapsule may then be removed.

Once removed, the membranous tissue (e.g., lens capsule, inner limitingmembrane, or other eye tissue) may be maintained in vitro or preparedfor in vivo transplantation. Membranous tissue is then placed on aglass, plastic, or polymer substrate. The glass substrate may be, forexample, a glass cover slip. The plastic substrate may be, for example,a tissue culture dish. The polymer substrate, for example, may be abiodegradable polymer. Biodegradable polymer films may includepoly-lactic acid, poly-glycolic acid, poly-lactic acid glycolic acidcopolymers (PLGA), including PLGA (50:50 lactic to glycolic acidcopolymer), poly-lactic acid polymers (PLLA), or polyethyleneglycol/polylactic acid copolymer (PEG/PLA), polyorthoesters,polyanhydrides, polyphosphazines and blends and copolymers thereof.Methods for using biodegradable polymer films may be found in, e.g.,U.S. Pat. No. 5,512,600 to Mikos et al.

For example, the methods discussed in U.S. Pat. No. 5,512,600 and in J.Biomedical Materials Research, Vol 34:87-93 (1997) by Giordano et al.may be used to maintain healthy lens capsule, inner limiting membrane,or other membranous tissue in vitro and in vivo. Biodegradable (e.g.,dissolvable after placement in an animal) polymer films comprisingpoly-lactic acid polymers (PLLA), poly-glycolic acid polymers,polyorthoesters, polyanhydrides, polyphosphazines, poly-lactic acidglycolic acid copolymers (PLGA), including PLGA (50:50 lactic toglycolic acid copolymer), and polyethylene glycol/polylactic acidcopolymer (PEG/PLA) films may be placed on the bottom of plastic petridishes. The lens capsule or other membranous tissue is then placed ontothe surface and smoothed down with the use of a pipette. The membranoustissue and polymer film are transplanted together. The film dissolves invivo leaving the membranous tissue behind. The film provides a greaterease of manipulation for the membranous tissue; for example, polymerfilms prevent lens capsule from curling, which is a problem observedwith prior art methods. In addition, further treatment of the membranoustissue may be applied following these steps.

Lens capsule tissue (or other membranous tissue) may be placed in anenvironment suitable for cell growth, such as a tissue culture incubatoror environmental chamber. In one embodiment, lens capsule tissue isimmersed in a phosphate buffered saline solution (PBS) and maintained at37° C. in a 95% O₂-5% CO₂ atmosphere. Following incubation, the PBS isremoved with a sterile pipette and the lens capsule is allowed to lieflat on the bottom of a sterile petri dish. The lens capsule is thensoaked in trypsin-EDTA for 1 hour to remove any lens epithelial cellsand subsequently, penicillin/streptomycin for 30 minutes for sterility.The lens capsules are then rinsed three times in PBS followed by threerinses in distilled water. Each rinse is performed carefully withsterile pipettes. Finally, the lens capsule and the petri dish it restson are sterilized under UV light for at least three hours.

In another embodiment, an interface chamber is used, wherein lenscapsule tissue (or other membranous tissue) is placed on wetted filterpaper covering a dish filled with phosphate buffered saline, andmaintained at 37° C. in a 95% O₂-5% CO₂ atmosphere. It will beunderstood that various saline solutions known in the art, such asbicarbonate-buffered saline, or other saline solutions, may besubstituted for PBS. Alternatively, culture medium (such as, forexample, those as RPM1, DMEM or Hamm's F12 (Life Technologies, MD)) maybe added to or may replace the saline in the methods, and growthfactors, antibiotics, serum, and other materials may be added to thesaline or culture medium used in maintaining lens capsule tissue.

Methods for modifying tissues include bulk modification methods andsurface modification methods. Bulk modification methods include methodswhere substantial portions of the tissue, not limited to surfaceportions of the tissue, are modified by the method. Surface modificationmethods include methods wherein the tissue is modified at and near tothe surface of the tissue, but is not greatly modified in other portionsof the tissue.

The methods of the invention as applied to lens capsule tissue, whetherbulk or surface modification methods, include removal of a lens capsulefrom an eye, flattening the lens capsule onto on a sterile glass orplastic substrate, such as a culture dish, microscope slide or a glasscoverslip, that is submersed in phosphate buffered saline or othersuitable solution, followed by further treatment of the lens capsule. Itwill be understood that similar treatments may be applied to innerlimiting membrane tissue, Bruch's membrane, amniotic membrane, or othertissue.

Plastic substrates such as culture dishes and glass substrates such asmicrosope slides may be sterilized by standard procedures, such as byirradiation with ultraviolet light, immersion in acid followed byrepeated washing in sterile distilled water, or other procedures knownin the art. In addition, plastic or glass substrates may be used with orwithout surface coatings. Surface coatings may include collagen,collagen gel, fibronectin, laminin, a silane coating such as polymethylsilane, a polymer coating such as poly-L-lysine, or other coating knownin the art.

In embodiments of the invention, the substrate is prepared for themembranous tissue. For example, tissue-culture plastic may be rinsed ina 70% ethanol solution to remove dust and oils and allowed to air dry.Following the drying step, the tissue culture plastic may be coveredwith a solution comprising a desired extracellular matrix molecule(e.g., 4 mg/ml collagen, type I rat tail in PBS, 1 μg/ml laminin fromhuman placenta in PBS, or 25 μg/ml fibronectin from human plasma in PBS)(collagen and fibronectin may be purchased from Sigma, St. Louis, Mo.).After one hour, the plastic may be rinsed in sterile distilled watertwice and allowed to dry under UV overnight. If the lens capsulesubstrates are not immediately stamped, they are stored at 4° C.

Bulk modification methods for modifying membranous tissue such as lenscapsule tissue include methods for modifying the thickness,permeability, and other properties of the lens capsule tissue. In oneembodiment of the bulk modification method, such further treatmentincludes use of an excimer laser to ablate the surface of the lenscapsule so that the overall thickness of the lens capsule is reduced.For example, the lens capsule may be ablated by a laser or ion beam, orby mechanical methods, so that the overall thickness mimics thethickness of Bruch's membrane.

A laser, such as an excimer laser (e.g., an argon fluoride laser (LambdaPhysik, Model 201 E)) may be used to provide pulses of laser lighteffective to ablate the surface of a lens capsule. For example, pulse ofbetween about 10 to 20 ns duration, delivered at a frequency of about 1to 50 Hz, with pulse energy densities of between about 300 to 500millijoules per square centimeter (mJ/cm²) are effective to ablate thesurface of a lens capsule in a desired manner. Each pulse is effectiveto ablate the tissue to a depth of between about 5 to 50 microns.Accordingly, repeated pulses are effective to reduce the thickness ofthe lens capsule tissue to a desired overall thickness. Methods as havebeen applied to the cornea may be followed or adapted and are suitablefor use in photoablation of lens capsule tissue. Such methods of cornealphotoablation are disclosed in, e.g., U.S. Pat. No. 4,665,913 toL'Esperance, U.S. Pat. No. 5,634,920 to Hohla, and U.S. Pat. No.5,735,843 to Trokel.

In another embodiment of the bulk modification method, such furthertreatment following placement of tissue on a glass substrate includesuse of a laser, such as, e.g., a YAG laser to produce micropores in thelens capsule. Such bulk modification by providing micropores alters theproperties of the lens capsule tissue so as to provide a more favorablesubstrate for cell attachment and alters the biochemical properties ofthe lens capsule tissue to allow more ready exchange of fluid andsolutes across the tissue. In embodiments of the invention, themicropores are sized on the order of 10s of nanometers (nm) or less indiameter. Thus, micropores produced by the bulk modification methods mayrange in size between about 0.01 micron to about 10 microns, preferablybetween about 0.1 micron to about 1 microns. An erbium YAG laser can beused to provide pulses of between about 10 to 50 ns duration, at energylevels of between about 1 to 50 mJ, preferably between about 1 to about20 mJ, effective to ablate holes in lens capsule tissue according to themethods of the invention.

In another embodiment of the bulk modification method, such furthertreatment following placement of membranous tissue on a glass substrateincludes use of an ion beam to produce micropores in the lens capsule toprovide a more favorable substrate for cell attachment and to allow moreready exchange of fluid and solutes across the tissue. See, for example,Goplani et al. J Membr. Sci 118:93-98 (2000), Xu et al., in MaterialResearch Society Symposium Proceeding Vol. 540 “MicrostructuralProcesses in Irradiated Materials”, pages 255-260 (1999), and Ohmichi etal., J. Nuclear Materials 248:354-359 (1997). In embodiments of theinvention, the micropores are sized on the order of 10s of nms to a fewTm in diameter.

The membranous tissue may be freeze dried for purposes of exposing tothe ion beams. Alternatively, the membranous tissue may be dried outentirely, then rehydrated after the micropores are made. An ion beam,such as a 120 MeV beam of Si²⁸ ions, may be used to irradiate thetissues. Following exposure to the ion beam, the membranous tissues maybe rehydrated. Biological etching using collagenase and other proteasesor proteolytic enzymes, as discussed below, may be used to enlarge themicroholes if larger holes are desired.

In another embodiment of the bulk modification method, treatment of themembranous tissue includes deposition of proteolytic enzymes onto themembranous tissue effective to biologically etch the surface andinterior of the membranous tissue to provide desired topology andsurface adhesion properties to the tissue. In some embodiments of thismethod, the deposition step includes contacting the lens capsule orother membranous tissue with a microcontact printing stamp carryingenzymes effective to biologically etch the surface and interior of thetissue. After stamping of the enzymes onto the tissue, albumin or anenzyme inhibitor may be used to stop the reaction after a given time.For example, incubation with collagenase is preferentially carried outfor various periods up to 26 h at 20° C. in a constant temperature waterbath, and the collagenase reaction stopped by the addition of EDTA to afinal concentration of 50 mM. Incubation with trypsin (e.g., 0.25%trypsin in a balanced salt solution without calcium or magnesium) may beperformed at about 0 to 5° C. for about 6 to about 18 hours. Followingthis incubation with trypsin, the trypsin solution may be removed andthe membranous tissue incubated at 37° C. for 20 to 30 minutes beforewashing with a wash solution containing divalent cations (such ascalcium and magnesium) in the amount of about 1 to about 5 mM (andoptionally containing a trypsin inhibitor such as soybean trypsininhibitor). Alternatively, membranous tissues may be incubated withdispase (about 0.5 to about 3 U/ml) or other proteolytic enzymes in abalanced salt solution that is substantially divalent cation-free at 37°C. for up to several hours before removal of the solution and washing ofthe membranous tissue with a balanced salt solution containing about 1to about 5 mM divalent cations.

In embodiments of the bulk modification methods, for example, agentssuch as collagenase, trypsin, chymotryptsin, dispase, liberase,thermolysin, pepsin, papain, and other proteases may be applied assolutions in distilled water, phosphate-buffered saline, or otherbuffered solution, at concentrations ranging between about 0.01 mg/mL toabout 100 mg/mL, preferably between about 1 mg/mL to about 20 mg/mL, tothe surface of a microcontact printing stamp. The surface of the tissue,such as lens capsule tissue, may be contacted in air or while immersedin a saline solution. Where the protease is active in the absence ofcalcium, such as for trypsin, chelating agents such as EDTA and EGTA,preferably at concentrations in the range of between about 1 to about 10mM, may be included in the solutions. In such cases, enzymatic actionmay be halted when desired by the addition of calcium and or magnesiumto the solution. In any case, enzymatic action may be stopped bydilution with excess of enzyme-free solution or by addition of anappropriate enzyme inhibitor. (For example, trypsin may be inhibited bya trypsin inhibitor such as soybean trypsin inhibitor (T-9003, SigmaChemical Co. St. Louis, Mo.)).

In another embodiment of the bulk modification method, treatment ofinner limiting membrane or lens capsule tissue includes impregnation ofthe tissue with a deactivated enzyme, such as a deactivated collagenaseenzyme, that is activated by laser light illumination. For example, inone embodiment very small regions sized less than a micron in diameterof tissue are activated by illumination with a 2-photon confocal lasersystem. Enzymes activated in this way are effective to degrade orotherwise alter tissue in the small region where activation occurs,while nearby regions not activated by the confocal laser system remainunaltered. The activated enzyme may be flushed out or deactivated bywater. Enzymes suitable for the practice of the invention include butare not limited to collagenase, trypsin, chymotrypsin, dispase,liberase, papain, pepsin, thermolysin, and other proteases.

In one embodiment of the surface modification method, microcontactprinting techniques are used to fabricate chemical micropatterns ofbiomolecules onto tissue. For example, surface modification of lenscapsule tissue may include deposition of patterns of biomolecules ontolens capsule tissue. Such patterns may include repeated iterations ofgeometric or linear patterns, or may include only a few, or a single,pattern not made up of smaller pattern units. Thus, patterns of surfacemodification may include linear arrays of biomolecules deposited onto atissue surface, or curved arrangements of biomolecules, series ofcircularly-shaped patterns, such as rings or dots, of biomolecules, or aseries of other shapes, including multiple shapes in a single pattern,of biomolecules. Alternatively, such patterns may include extended areassubstantially covered by deposited biomolecules, or extended areassubstantially devoid of deposited biomolecules. It will be understoodthat the methods include any suitable pattern comprising lines, shapes,or regions of deposited molecules, including regions devoid of depositedmolecules situated between regions with deposited biomolecules. Suchmicropatterns may, in general improve cell attachment and growth on themodified membranous surface. However, in embodiments of the invention,micropatterns are produces where regions of the modified membranoustissue are rendered less suitable, or unsuitable, for cell attachmentand growth. In this way, cell attachment and growth may be directed toand limited to those regions of the membranous tissue that have not beenso treated.

Microcontact printing stamps may include the entire pattern to bedeposited onto target tissue, or may include a portion of the desiredpattern. Where the stamp includes a portion of the desired pattern,multiple applications of the microcontact printing stamp to the tissuesurface are effective to provide a desired pattern of biomolecules onthe tissue surface. Where the stamp includes the entire pattern,biomolecules may be deposited onto the microcontact printing stampitself in the desired pattern.

The patterns of biomolecules on a microcontact printing stamp may bedetermined by directed placement of the biomolecules on the stamp, ormay be determined by the surface geometry of the stamp. Where thepattern of biomolecules is determined by the surface geometry of thestamp, the geometric pattern may include locally-raised ridges, wherecontact of the stamp with a source of biomolecules is effective todeposit such biomolecules onto the raised surfaces, with substantiallyno biomolecules being deposited on other, non-raised portions of thesurface. In such a microcontact stamp, the pattern of biomoleculesdeposited onto a tissue would follow the pattern of the raised surfacesAlternatively, the pattern may include depressions, valleys or fissures,such as scratches made into a surface, where contact of the stamp with asource of biomolecules is effective to deposit such biomolecules onto amajor portion of the surface, with substantially no biomolecules beingdeposited on the depressed portions of the surface. In such amicrocontact stamp with depressions, biomolecules would be depositedover a substantial portion of the tissue, with regions substantiallylacking deposited biomolecules following the pattern of the depressedsurfaces.

In some embodiments of this method, the patterns are sized on the orderof a few microns or less. Accordingly, in embodiments of the surfacemodification methods of the invention, the individual patterns of whichthe overall patterns are comprised may range in size between about 0.1micron to about 20 microns, preferably between about 0.5 microns toabout 5 microns.

Biomolecules suitable for deposition onto tissue surface includeproteins, peptides, organic molecules, oligosaccharides, and small chainpolymers, including but not limited to collagen, hyaluronic acid,keratin sulfate, glycosaminoglycan, methylacrylate, poly (methylmethacrylate), polystyrene, poly(methyl styrene), polylysine, polylacticglycolic acid (PLGA)-derivatized polylysine, polylysine peptides, andsilane polymers such as octadecyltrichlorosilane (OTS). Surfacemodification comprising deposition of biomolecules is effective to alterbiological properties of the tissue, such as the ability or ease ofattachment by cells placed onto microfabricated tissues. For example,deposition of hydrophobic molecules is effective to deactivate selectivecell attachment sites on lens capsule tissue.

Microcontact printing stamps may be made of any material capable ofretaining a suitable pattern, such as glass, ceramic, metal, plastic,polymer, or other material. In presently preferred embodiments of themethod, microcontact printing stamps include poly(dimethylsiloxane)(PDMS), which is commercially available (e.g., Sylgard 184 from DowCorning, Midland Mich. 48640). Microcontact printing stamps may be castin PDMS from masters containing desired patterns, such as, for example,a grid pattern of lines. Alternatively, where the pattern to be formedis determined by the pattern of deposition of biomolecules onto atissue, the stamp may include a simple surface, such as a flat surface,suitable for carrying biomolecules. Such stamps may include pins,slotted pins, bars or rods, for example, and may have circular,triangular, square, rectangular, other polygonal or irregularly shapedperimeters.

In embodiments of the surface modification method, the surface of thelens capsule tissue is masked to cover part, but not all, of the surfaceof the lens capsule tissue, and then irradiated with ultraviolet (UV)radiation effective to denature the extracellular matrix (ECM) of theexposed portions of tissue. This deactivates molecules specific for celladhesion, and to inhibits or prevents cell adhesion and growth in theexposed, but not the covered, regions. Thus, in this embodiment of themethods of the invention, portions of the substrate are renderedunsuitable for cell attachment and growth. In this way, growing cellscan be directed to desired regions, and away from undesired regions.

In embodiments of the invention, the entire substrate surface may bedeactivated to prevent attachment or growth of cells, and then specificregions reactivated. By deactivating proteins that are specific forcellular adhesion, the growth of cells may be limited to confinedregions. A deactivating substance is one that prevents the attachment,the spread, or both, of growing cells. For example, 0.2% polyvinylalcohol (PVA) solution and mucilage are effective deactivatingsubstances.

A surface may be deactivated, and a portion of that surface reactivated,by application of a deactivating substance to the surface. For example,0.2% PVA applied to the surface of the lens capsule is effective todeactivate the surface of the lens capsule. Exposure of the deactivatedlens capsule surface to a micropattern of light from an excimer laser iseffective to ablate a micropattern on the lens capsule surface. Forexample, a micropattern may be produced on the lens capsule surface byillumination of the lens capsule surface through an irradiation mask.The ablated micropattern, by removing or altering the deactivatingsubstance, reactivates portions of the substrate to allow cell growthand spreading into the ablated regions, thereby directing cell growth tofollow a desired pattern.

The masking step may include placement of a grid onto the tissue, wherethe grid includes a material effective to prevent irradiation of thesurface by a source of radiation, such as UV radiation. The grid may bemade of materials including metal, glass, plastic, ceramic, polymer,protein, or other material effective to absorb or reflect UV radiation.

In an alternative embodiment of the masking method, the masking stepincludes using microcontact printing techniques to apply a pattern ofprotecting molecules onto the surface of the lens capsule tissueeffective to prevent ECM denaturation in regions covered by theprotecting molecules. Thus, the grid of a masking step may include acoating on the surface effective to screen the surface from irradiation.Such a coating may include a protein, preferably one rich in tyrosineand other amino acid residues that absorb ultraviolet light, a polymereffective to absorb UV light, or a small molecule effective to screen UVlight, such as, for example, para-amino benzoic acid (PABA).

It will be understood by one of skill in the art that surfacemodification methods and bulk modification methods may each be appliedto a single tissue. Thus, for example, the same lens capsule tissue maybe treated with both surface modification and bulk modification methodseffective to provide microfabricated lens capsule tissue.

Microfabricated tissues are suitable substrates for growing cells. Amethod for growing cells on microfabricated tissues includes providing amicrofabricated tissue produced by one of the methods described above,and applying cells to the microfabricated tissue. For example, themicrofabricated tissue may include a microfabricated lens capsule with apattern on its surface, such as a pattern of collagen, and the cells mayinclude IPE cells, RPE cells, stem cells, or other cells. In preferredembodiments of the invention that includes autologous tissue and cells,the microfabricated tissues and the cells are obtained from the sameanimal.

The invention also provides methods for using microfabricated tissues,including surgical methods for transplanting microfabricated tissuesinto an animal. In preferred embodiments, the methods for transplantingmicrofabricated tissues into an animal include surgical methods fortransplanting microfabricated tissues into the eye of an animal. In mostpreferred methods, the transplantation of microfabricated tissues intothe eye of an animal includes transplantation of microfabricated lenscapsule tissue near to or into the retina of an animal. In someembodiments, the transplanted tissue further includes cells grown onmicrofabricated lens capsule tissues. In other embodiments, thetransplanted tissue includes RPE cells, IPE cells, stem cells, or othercells grown on microfabricated lens capsule tissues. Alternatively,dissolvable polymer substrates may be used for growing cells fortransplantation. In further embodiments, the transplanted tissueincludes RPE cells, IPE cells, stem cells, or other cells grown onmicrofabricated membranous tissues or on dissolvable polymer substrates,where the cells and tissues are taken from the same animal as the animalinto which they are transplanted (autologous tissue).

Methods for isolating or removing RPE cells from an eye may be found inPfeffer, B. A., Chapter 10, “Improved Methodology for Cell Culture ofHuman and Monkey Retinal Pigment Epithelium,” Progress in RetinalResearch, Vol. 10 (1991) Ed. Osborn, N., and Chader, J.; these methodsmay also be applied to IPE cells. The cells may be removed from a donoreye, or from the intact eye of a patient, including the eye that willultimately receive a transplant of microfabricated tissue with cells.Methods for harvesting cells obtained in a biopsy, as for an autologoustransplantation procedure, may be found in Lane, C., et al. Eye 3:27-32(1989). Further methods for procurement of RPE and IPE may be found,e.g., in Abe et al., 1999, Thumann, et al., 1999; Lappas et al., 2000;and in Thurmann et al., 2000.

The IPE cells, RPE cells, stem cells, or other cells may be dispersed insaline, such as phosphate-buffered saline, at a density of between about10⁴ cells/mL to about 10⁷ cells/mL. Isolated RPE cells, IPE cells, stemcells or other cells may be applied to microfabricated tissue, forexample, to microfabricated lens capsule tissue by gently pipetting asolution containing IPE cells, RPE cells, stem cells or other cells ontothe microfabricated tissue immersed in PBS, followed by maintenance ofthe cells and tissue at 37° C. in a sterile 95% O₂-5% CO₂ atmosphere for12 hours. The PBS may be removed with a sterile pipette and the lenscapsule allowed to lie flat on the bottom of a sterile petri dish orother container. The lens capsule may then be soaked in trypsin-EDTA for1 hour to remove any lens epithelial cells and subsequently,penicillin/streptomycin for 30 minutes for sterility. Following this,the lens capsules may then be rinsed three times in PBS followed bythree rinses in distilled water. Each rinse should be performedcarefully with sterile pipettes. Finally, the lens capsule and itssupport are sterilized under UV light for at least three hours.

Before the application of cells, i.e. either in situ and/or in vivothrough transplantation, microfabricated tissues, such as lens capsule,inner limiting membrane, Bruch's membrane, and other tissues, may bemodified and microfabricated as described above. Alternatively, or inaddition to such modification and microfabrication, a microfluidicchannel or pattern of microfluidic channels may be placed onto amembrane surface to be modified, and a suspension of cells or moleculesmay be delivered to the membrane surface. For example, a microfluidicnetwork as described by Delamarche et al. (Science 276:779-781 (1997)),herein incorporated by reference in its entirety, may be applied to amembrane surface in order to modify the membrane. In such a procedure, atrough or series of troughs may be formed in PDMS or other biocompatiblematerial, the troughs configured to form conduits upon placement of thePDMS onto a membrane surface, with the membrane surface serving as aconduit wall. Cells or biomolecules may be brought into contact with themembrane surface by flowing a solution containing the cells orbiomolecules, or containing both cells and biomolecules, through theconduits. The cells and biomolecules may thus be deposited onto, or mayotherwise modify, the exposed surface of the membrane that forms a wallof the conduit.

Isolated RPE cells, IPE cells, stem cells, or other cells may also beapplied to a membranous tissue which has been partially covered by astencil. A stencil suitable for the practice of the invention isconfigured with a pattern of holes or passages passing through itssurface. Such a stencil covers underlying membranous tissue when thestencil is applied to a membranous tissue, while the pattern of holes orpassages is effective to leave portions of underlying membranous tissueexposed. A stencil for microfabricating tissue may have a rim thickerthan the bulk of the stencil in order to help provide mechanicalstrength. A stencil having such a pattern of holes or passageways may beapplied to a surface of membranous tissue to be microfabricated,effective to direct the growth of cells on the membranous tissue or tomodulate the exposure of the membranous tissue to external agents andtreatments. In some embodiments of this method, the patterns may besized on the order of a few microns or less, or on the order of severalmicrons, so that patterns may range in size between about 0.1 micron toabout 100 microns, preferably between about 1 micron to about 75microns, more preferably between about 5 microns to about 50 microns. Astencil may be formed of any suitable biocompatible material, such as,for example, PDMS or PLGA/PEG copolymer. A suitable stencil material maybe solid or gelatinous, and is preferably flexible. A stencil materialmay also be biodegradable (e.g., PLGA/PEG copolymer). Stencils suitablefor application to membranous tissue for transplantation into the eye ofan animal may be made by methods similar to those described in, forexample, Folch et al. J. Biomed. Mater. Res. 52:346-353 (2000), herebyincorporated by reference herein in its entirety.

Transplantation of microfabricated lens capsule tissue into thesubretinal space may be effected by any means providing access to thesubretinal space. Access to the subretinal space may be provided, forexample, by a scleral incision placed laterally on the eye, or via thevitreous humor by a more frontal incision. Procedures providing accessto, and transplantation into, the retina, including the subretinalspace, have been described; see, for example, Abe et al., Tohoku J. Exp.Med. 189:295-305 (1999), Abe et al., Tohoku J. Exp. Med. 191:7-20(2000), Lappas et al., Graefes's Arch Clin Exp Ophthalmol. 238:631-641(2000), Thumann, et al., Arch. Ophthalmol. 118:1350-1355 (2000), U.S.Pat. No. 5,962,027 to Hughes and U.S. Pat. No. 6,045,791 to Liu.

Alternatively, a microcontact printing stamp, or a stencil, may beapplied to an ocular membrane in vivo. For example, access to Bruch'smembrane within an intact, living eye may be accomplished by standardsurgical procedures, including formation of a bleb by infusion of a gas,saline, mineral oil, or other biocompatible liquid into the subretinalspace of an eye, and placement of a microcontact printing stamp or of astencil onto Bruch's membrane. Such application of a microcontactprinting stamp, or of a stencil, may be performed wet, that is in thepresence of normal bodily fluids, PBS or other artificial physiologicalsolution, mineral oil, or other biocompatible liquid. Alternatively,such application of a microcontact printing stamp, or of a stencil, maybe performed dry, that is in the absence of normal bodiy fluids orartificial physiological solution, by, for example, filling thesubretinal space with an inert gas such as nitrogen or argon.

An intact Bruch's membrane may be prepared for in situ microfabricationby scraping or otherwise debriding Bruch's membrane to remove RPE cellsbefore application of a stamp or a stencil. A stamp or stencil may thenbe used to provide a desired pattern onto a surface of the membrane. APDMS stencil, for example, having a pattern of passages may be appliedto the surface of Bruch's membrane; Cells are able to attach and grow onthe membrane surfaces exposed by the passages. Similarly, biomoleculesin a solution on contact with the membrane surface are able to contactand modify or adhere to the membrane surfaces exposed by the passages.The pattern of passages is effective to provide a pattern suitable fordirecting the depostion of biomolecules, and of directing the growth ofadded cells, such as RPE, IPE, stem cells, or other added cells, or theregrowth of endogenous cells from other regions of the eye.Alternatively, or in addition, a microcontact printing stamp carryinglaminin, fibronectin, or other desired coating agent, may be applied tothe ocular membrane surface. In further alternative methods embodyingfeatures of the invention, internal ocular membranes may be accessed viathe sclera, as, for example, by scleral puncture, scleral incision,formation of a scleral window, or other method. In methods takingadvantage of scleral access, there may be no need to traverse thevitreous humor in order to access ocular membranes for treatment.

EXAMPLE 1

Microcontact printing was used to deposit micron-sized patterns ofbiomolecules onto lens capsule tissue. Poly(dimethyl siloxane) (PDMS)stamps were cast from masters containing a topological pattern of gridlines spaced 50 microns apart. The PDMS stamp was made from a masterthat was microfabricated from a silicon wafer. PDMS stamps were used tomicrofabricate patterns onto lens capsule tissue. Shown in FIG. 2 is ascanning electron micrograph (SEM) of a PDMS stamp used to deposit amicropattern onto a piece of human lens capsule tissue.

The PDMS stamp shown in FIG. 2 has a surface topology given by ahexagonal array of 5 μm-wide lines. Each line is separated byapproximately 50 μm. FIG. 3 shows a human lens capsule stamped with thePDMS stamp shown in FIG. 2. The PDMS stamp was used to deposit hexagonalpatterns of a PVA and fluorescein solution (2% PVA and 0.1 mg/mLfluorescein) onto the lens capsule. This example shows that the stamp iseffective to produce a pattern on a surface corresponding to the patternof the stamp.

EXAMPLE 2

A SEM of a PDMS stamp with circular patterns used for micropatterningtissue is shown in FIG. 4. As shown, the stamp has a surface topologygiven by an array of circular wells of approximately 50 μm in diameter.When the relief pattern is coated with an inhibitory molecule, such asPVA or mucilage, and the stamp applied to a lens capsule, the inhibitorymolecules are transferred to the lens capsule in the pattern shown. FIG.5 shows the surface of a lens capsule that has been patterned with aPDMS stamp having a pattern as shown in FIG. 2 and RPE cells grown onit. This example shows that the stamp is effective to place a pattern onthe lens capsule surface that corresponds to the pattern of the stamp,and for cell growth to be patterned according to the pattern of thestamp.

Thus, application of the stamps of the invention are able to depositinhibitory molecules in patterns that can direct the growth of cellsgrowing on a patterned substrate. Because the lens capsule activelyallows growth, patterns of inhibitory molecules, such as PVA, arepreferred for patterned growth. Use of the stamp on substrates treatedto inhibit growth would require the use of activating molecules topattern growth on the substrate.

EXAMPLE 3

Masking of the surface of lens capsule tissue and then irradiating theexposed surface, but not the masked surface, with UV radiation isaccomplished by placement of a SEM grid onto the surface of lens capsuletissue. A SEM grid with spacing of 50 microns is placed onto the exposedsurface of an excised lens capsule tissue resting on a glass coverslipimmersed in phosphate-buffered saline. The surface of the lens capsuletissue and the SEM grid are not immersed in the phosphate-bufferedsaline, but rise above the level of the phosphate-buffered saline. UVlight is directed onto the exposed surface of the lens capsule tissueeffective to irradiate the lens capsule tissue not resting immediatelybelow the SEM grid material. After irradiation, the SEM grid is removed.The lens capsule surface has a micropattern of lines including tissuenot irradiated (regions under SEM grid material) enclosing regionscomprising irradiated tissue.

EXAMPLE 4

Growth of monolayer cultures of retinal pigment epithelium and irispigment epithelium cells is facilitated by flat substrate and byinsuring that the substrate does not curl or fold upon implantation inthe subretinal space or other region of the eye. A biodegradable matrixcoating was found to prevent folding and curling of lens capsule tissue.Such a biodegradable matrix coating, which prevents substrate curling orfolding is suitable for use as a substrate for the growth of monolayercultures of retinal pigment epithelium and iris pigment epithelium cellsfor implantation into an eye.

A biodegradable polymer matrix of poly(dl-lactide/glycolide) was made bydissolving 50 mg of a 90:10 mixture of poly(dl-lactide/glycolide)(Polysciences, Inc., Warrington Pa. 18976) in different amounts ofdichloromethane to make solutions of 100 mg/mL (0.5 mL dichloromethane),150 mg/mL (0.33 mL dichloromethane), and 200 mg/mL (0.25 mLdichloromethane).

Human lens capsules obtained during cataract surgery were stored inphosphate buffered saline at 4° C. prior to sterilization underultraviolet light (254 nm for three hours) and treatment with 0/05%trypsin-(ethylene diamine tetraacetic acid) for ten minutes at 37° C. toremove native epithelial cells. Treated lens capsules were spread in asingle layer on Parafilm® (Pechiney Plastic Packaging, Inc., Neenah,Wis. 54956) in a Petri dish and coated on one side with thepoly-d-lactyl glycolic acid (PLGA) biodegradable matrix of a singleconcentration by dispensing 5 μL, 10 μL or 20 μL of the PLGA solutionfrom a pipette onto the lens capsule surface. The PLGA solution wasallowed to spread over a 5 mm diameter circular area containing theflattened lens capsule. The solvent was evaporated in a chemical hood.

Five New Zealand White rabbits weighing 2.5 to 3.5 kg underwentimplantation of the lens capsule/PLGA complex following ketamine (40mg/kg) and Xylazine (5 mg/kg) anesthesia. Tropicamide 0.5% andPhenylephrine 2.5% eyedrops were instilled into the conjunctival sac ofthe left eye every five minutes for three doses. Standard three-portpars plana vitrectomy was performed, and a retinal bleb was inflated inthe macular area by injection of approximately 0.5 mL of balanced saltsolution through a 42-gauge needle. A retinotomy 1 mm in diameter wascreated, and the lens capsule/PLGA was inserted into the subretinalspace through the retinotomy with subretinal forceps. The retina wasthen reattached by air-fluid exchange.

The operated eyes were removed one week after implantation of the lenscapsule/PLGA and fixed in 1.25% glutaraldehyde/1% paraformaldehyde incacodylate bufer (pH 7.4). The eyes were then cut open, fixed,post-fixed in osmium tetroxide, dehydrated in a graded series ofethanol, embedded in epoxy resin, cut into 1 μm sections and stainedwith toluidine blue.

FIG. 6 shows a photomicrograph of a microfabricated lens capsule on apolylactide/polyglycolide carrier matrix. Histological studies performedone week post-implantation demonstrated that the lens capsule remainedflat on Bruch's membrane. This is illustrated by a section of a rabbitretina having a lens capsule/PLGA implant taken 1 week afterimplantation is shown in FIG. 7. There was local disruption of thephotoreceptor layer, and an overlaying rtinal detachement, presumably inthe area previously occupied by the bleb. The PLGA was almost completelydissolved in all cases. The five implantations demonstrated that lenscapsule coated with PLGA was easy to handle during surgery, and hadsufficient rigidity so that the implants remained flat within thesubretinal space. No evidence of significant inflammatory reaction wasnoted.

This example shows that PLGA greatly improves the surgical handling oflens capsule during subretinal implantation and allows the lens capsuleto be implanted without curling. While untreated lens capsule may rollinto multiple layers or fold during implantation, lens capsule treatedwith a bioabsorbable matrix coating such as the PLGA coating used inthis example remains relatively flat in the subretinal space afterimplantation. Because the PLGA degrades within a few weeks, concerns forlate immune reactions to an implant are allayed. This exampledemonstrating improved mechanical characteristics of coatedmicrofabricated lens capsule illustrates that coating microfabricatedtissues such as lens capsule, inner limiting membrane, Bruch's membrane,and other tissues, overcomes the limitations of the mechanical weaknessof the untreated tissue and provides an improved substrate forimplantation of tissues and cells.

EXAMPLE 5

One therapy for AMD is to transplant suspensions of either RPE cells orIPE cells to rescue the diseased retina. The present invention providesnovel tissue engineering techniques to precision engineer autologoushuman tissues as a substrate for transplanting cells, such as IPE cells,RPE cells, stem cells, and other cells. Suitable tissues includemembranous tissues, such as lens capsule (e.g., human lens capsule),inner limiting membrane tissue, Bruch's membrane tissue, corneal tissue,amniotic membrane tissue, serosal membrane tissue, mucosal membranetissue, and neurological tissue.

A microgeometry of inhibitory molecules is arranged onto the surface ofa suitable substrate. Suitable substrates include human lens capsule,collagen gel, collagen-, fibronectin-, and laminin-coated plastic, and adissolvable polymer such as PLGA or PLLA. Human lens capsules may beobtained during cataract surgery. Cultures of experimental RPE cells aregrown on these microengineered surfaces and analyzed using scanningelectron microscopy, atomic force microscopy, and fluorescencemicroscopy. Comparisons between microfabricated surfaces of autologoustissue and synthetic surfaces and membranes are then made.

These comparisons demonstrate that individual RPE cells may be directedto grow in microenvironments on the respective biological surfaces.Although all surfaces studied are amenable to micromachining, includinghuman lens capsule, it will be understood that different tissueengineering methods may be used to vary cell-to-cell distance and themicroenvironment of growth factors and cell adhesion molecules.

EXAMPLE 6

In this example, isolated RPE cells are applied to a lens capsulemembrane tissue which has been partially covered by a stencil and theirgrowth on the lens capsule is directed by the stencil pattern.Fibronectin (from human plasma, 25 μg/ml in PBS) is coated onto anexcised lens capsule tissue resting on a glass coverslip immersed inphosphate-buffered saline. A PDMS stencil having a regular pattern ofhexagonal holes of about 50 μm across, the holes being spaced about 10μm apart, is sterilely placed onto the excised lens capsule tissue. Thefibronectin promotes the adherence of the stencil to the lens capsule aswell as promoting the adherence of added cells. RPE cells dispersed incell growth medium (10⁷ cells/mL) are sterilely added to the salinesolution onto the surface of the lens capsule that is partially coveredby the stencil. The lens capsule, stencil, glass coverslip, added cells,and cell growth medium are maintained in a tissue culture dish in atissue culture incubator and are maintained under suitable cultureconditions at approximately 37° C. in a 95% O₂-5% CO₂ atmosphere. Thestencil's pattern of hexagonal holes leaves portions of underlyingmembranous tissue exposed to the RPE cells. The RPE cells adhere to theexposed lens capsule tissue, and grow on it in hexagonal patternsdirected by the stencil. The resulting microfabricated lens capsuletissue with adherent RPE cells is suitable for transplantation into theeye of an animal.

EXAMPLE 7

In this example, a microcontact printing stamp is applied to an ocularmembrane in vivo, providing a microfabricated membranous surfacesuitable for growth of cells. Such a microfabricated membranous surfacesuitable for the growth of cells aids in the treatment of eye diseasesor conditions stemming from defects of ocular membranes or ocular cells.For example, in an eye having a region of diseased RPE cells, or RPEcells which are not functioning properly, removal of the defective RPEcells and microfabrication of the underlying Bruch's membrane,optionally with the addition of cells, is effective to treat the eye.

A subretinal bleb is used to access a retinal region in an eye of apatient having diseased RPE cells. The bleb is formed by infusion ofsterile saline into the subretinal space following a 3-port pars planavitrectomy and puncture of a small pathway through the neural retina.RPE cells exposed by the bleb are removed from a portion of Bruch'smembrane by scraping or otherwise debriding Bruch's membrane, such as bytechniques used in choroidal neovascular surgery. A microcontactprinting stamp having a pattern of 50 μm-diameter circles spaced 10 μmapart is coated with 25 μg/ml fibronectin from human plasma in PBS,rolled into a tubular configuration, and inserted into a needle. Theneedle is connected to a syringe containing PBS. The microcontactprinting stamp and PBS are gently injected into the bleb within thesubretinal space, and the stamp unrolled by action of the needle and ofPBS delivered by the needle. The microcontact printing stamp is placedin contact with the exposed Bruch's membrane. Such placement of a coatedmicrocontact printing stamp is effective to deposit a pattern offibronectin onto the exposed Bruch's membrane and to prepare the intactBruch's membrane in situ for microfabrication effective to provide apattern suitable for directing the growth of added cells or the regrowthof endogenous cells from other regions of the eye. After 15 minutes, themicrocontact printing stamp is removed from the bleb via the pathwaythrough the neural retina. In alternative treatments, the microcontactprinting stamp is left in contact with Bruch's membrane for periodsvarying between about 1 minute up to about 1 hour. Following removal ofthe microcontact printing stamp, a dispersion of RPE cells in PBS (10⁶cells/mL) is gently infused into the bleb. In further alternativetreatments, in which the microcontact printing stamp is made ofbiodegradable materials, the microcontact printing stamp is not removed,but may remain in place as cells are added and afterwards. Theinstruments are then removed from the eye, the incisions are closed andstandard post-operative care is given to the patient. The RPE cellsproliferate to cover the exposed portion of Bruch's membrane and aid inmaintaining the health of Bruch's membrane and in the support ofoverlying neural retina.

EXAMPLE 8

In this example, a stencil is placed onto an ocular membrane in vivo,providing a microfabricated membranous surface suitable for growth ofcells. A subretinal bleb is used to access a retinal region in an eye ofa patient having diseased RPE cells. The bleb is formed by infusion ofsterile saline into the subretinal space following a 3-port pars planavitrectomy and puncture of a small pathway through the neural retina.RPE cells exposed by the bleb are removed from a portion of Bruch'smembrane by scraping or otherwise debriding Bruch's membrane, such as bytechniques used in choroidal neovascular surgery. Fibronectin (fromhuman plasma, 25 μg/ml in PBS) is diffused into the bleb to coat theexposed Bruch's membrane and to promote adherence of an added stencil. Astencil made from PDMS and having a pattern of hexagons measuring 60 μmacross at their widest dimension, and spaced 10 μm apart is rolled intoa tubular configuration in PBS and inserted into a needle. The stenciland PBS are gently injected into the bleb within the subretinal space,and the stencil unrolled by action of the needle and PBS delivered bythe needle. The stencil is placed onto the exposed Bruch's membrane.Such placement of a stencil onto Bruch's membrane in situ is effectiveto provide a pattern on the exposed Bruch's membrane effective to directthe growth of added cells or the regrowth of endogenous cells from otherregions of the eye. Following placement of the stencil, a dispersion ofIPE cells in PBS (10⁵ cells/mL) is gently infused into the bleb. Theinstruments are then removed from the eye, the incisions are closed andstandard post-operative care is given to the patient. The IPE cellsproliferate to cover the portions of Bruch's membrane exposed by thegaps of the stencil and aid in maintaining the health of Bruch'smembrane and in the support of overlying neural retina.

1. An ocular implant, comprising: (a) a bioabsorbable substrate; (b) amicrofabricated membranous tissue layer secured to said bioabsorbablesubstrate; and (c) cells on the surface of said microfabricatedmembranous tissue layer, said cells separated into regions on saidsurface by creating a pattern on said surface enclosing said regions forreceiving said cells.
 2. The ocular implant as set forth in claim 1,wherein said tissue of said microfabricated membranous tissue layer isselected from the group consisting of lens capsule, inner limitingmembrane, comeal tissue, Bruch's membrane tissue, amniotic membranetissue, serosal membrane tissue, mucosal membrane tissue andneurological tissue.
 3. The ocular implant as set forth in claim 1,wherein said cells are cells selected from the group consisting of IPEcells, RPE cells and stem cells.
 4. The ocular implant as set forth inclaim 1, wherein said cells are received on said surface in situ or invivo.
 5. The ocular implant as set forth in claim 1, wherein said cellson said microfabricated membranous tissue layer are separated by growthinhibitory barriers.
 6. The ocular implant as set forth in claim 1,wherein said cells are separated by a patterned stencil.
 7. The ocularimplant as set forth in claim 1, wherein said bioabsorbable substratecomprises a material selected from the group consisting of glass,collagen, glycosaminoglycans, chitosan; poly(hydroxyalkanoates),poly(α-hydroxy acids), polyglycolic acid (PGA), polylactic acid (PLA),polylactide-polyglycolide (PGA-PLA) mixtures, alloys and copolymers(PLGA), poly(dioxanones), poly(E-caprolactone); poly(ortho esters),poly(anhydrides), poly(phosphazenes), poly(amino acids), and othercompounds, polymers, copolymers, alloys, mixtures and combinationsthereof.
 8. The ocular implant as set forth in claim 1, wherein saidbioabsorbable substrate is formed of a material selected from the groupconsisting of poly-lactic acid, polyglycolic acid, polyorthoesters,polyanhydrides, polyphosphazines, poly-lactic acid glycolic acidcopolymers, polyethylene glycol/polylactic acid copolymers and blendsand copolymers thereof.
 9. The ocular implant as set forth in claim 1,wherein said microfabricated membranous tissue layer is about 2 to about5 micrometers in thickness.
 10. The ocular implant as set forth in claim1, wherein said microfabricated membranous tissue layer has microporesor pits.
 11. The ocular implant as set forth in claim 1, wherein saidmicrofabricated membranous tissue layer has a micropattern ofbiomolecules.
 12. The ocular implant as set forth in claim 11, whereinsaid biomolecules of said micropattern of said microfabricatedmembranous tissue layer are selected from the group consisting ofproteins, peptides, organic molecules, oligosaccharides, and small chainpolymers.
 13. The ocular implant as set forth in claim 11, wherein oneor more of said biomolecules of said micropattern of saidmicrofabricated membranous tissue layer are selected from the groupconsisting of poly (methyl methacrylate), polystyrene, poly (methylstyrene), collagen, keratin sulfate, hyaluronic acid, glycosaminoglycan,octadecyltrichlorosilane, silane polymers, polylysine, polylacticglycolic acid (PLGA)-derivatized polylysine and polylysine peptides. 14.The ocular implant as set forth in claim 1, wherein said ocular implantis in a subretinal space.
 15. The ocular implant as set forth in claim1, further comprising a microfluidic network placed onto saidmicrofabricated membranous tissue layer.
 16. An implant, comprising: (a)a polymeric substrate; (b) a membranous tissue layer secured to saidpolymeric substrate; and (c) an array of cells on the surface of saidmembranous tissue layer, wherein said cells are separated into regionson said surface defining said array by creating a pattern on saidsurface enclosing said regions for receiving said cells.
 17. The implantas set forth in claim 16, wherein said tissue of said microfabricatedmembranous tissue layer is selected from the group consisting of lenscapsule, inner limiting membrane, corneal tissue, Bruch's membranetissue, amniotic membrane tissue, serosal membrane tissue, mucosalmembrane tissue and neurological tissue.
 18. The implant as set forth inclaim 16, wherein said cells are cells selected from the groupconsisting of IPE cells, RPE cells and stem cells.
 19. The implant asset forth in claim 16, wherein said cells are received on said surfacein situ or in vivo.
 20. The implant as set forth in claim 16, whereinsaid cells on said microfabricated membranous tissue layer are separatedby growth inhibitory barriers.
 21. The implant as set forth in claim 16,wherein said cells on said membranous tissue layer are in apredetermined pattern as depicted in FIG. 3 or FIG.
 5. 22. The implantas set forth in claim 16, wherein said cells are separated by apatterned stencil.
 23. The implant as set forth in claim 16, whereinsaid bioabsorbable substrate comprises a material selected from thegroup consisting of glass, collagen, glycosaminoglycans, chitosan;poly(hydroxyalkanoates), poly(α-hydroxy acids), polyglycolic acid (PGA),polylactic acid (PLA), polylactide-polyglycolide (PGA-PLA) mixtures,alloys and copolymers (PLGA), poly(dioxanones), poly(E-caprolactone);poly(ortho esters), poly(anhydrides), poly(phosphazenes), poly(aminoacids), and other compounds, polymers, copolymers, alloys, mixtures andcombinations thereof.
 24. The implant as set forth in claim 16, whereinsaid polymeric substrate is formed of a material selected from the groupconsisting of poly-lactic acid, polyglycolic acid, polyorthoesters,polyanhydrides, polyphosphazines, poly-lactic acid glycolic acidcopolymers, polyethylene glycol/polylactic acid copolymers and blendsand copolymers thereof.
 25. The implant as set forth in claim 16,wherein said membranous tissue layer is about 2 to about 5 micrometersin thickness.
 26. The implant as set forth in claim 16, wherein saidmembranous tissue layer has micropores or pits.
 27. The implant as setforth in claim 16, wherein said membranous tissue layer has amicropattern of biomolecules.
 28. The implant as set forth in claim 27,wherein said biomolecules of said micropattern of said membranous tissuelayer are selected from the group consisting of proteins, peptides,organic molecules, oligosaccharides, and small chain polymers.
 29. Theimplant as set forth in claim 27, wherein one or more of saidbiomolecules of said micropattern of said membranous tissue layer areselected from the group consisting of poly (methyl methacrylate),polystyrene, poly (methyl styrene), collagen, keratin sulfate,hyaluronic acid, glycosaminoglycan, octadecyltrichlorosilane, silanepolymers, polylysine, polylactic glycolic acid (PLGA)-derivatizedpolylysine and polylysine peptides.
 30. The implant as set forth inclaim 16, wherein said implant is in a subretinal space.
 31. The implantas set forth in claim 16, further comprising a microfluidic networkplaced onto said microfabricated membranous tissue layer.